The long-term objective of this work is to understand the biochemistry of nuclear DNA replication. This extremely complicated process requires the coordinated activity of 3 DNA polymerases and multiple accessory proteins, and is a primary target for variety of cancer chemotherapeutics. Thus, a greater understanding of the mechanism of DNA replication and how it can be inhibited may help lead to the development of novel chemotherapeutic strategies for the effective treatment of cancer. The enzyme complex of primary interest is DNA polymerase alpha-primae. On single-stranded DNA, primate synthesizes RNA primers that are further elongated by pol alpha via dNTP polymerization. These reactions are essential for initiation of new strands of DNA, both for initiation of leading strand synthesis and synthesis of Okazaki fragments on the lagging strand. While many of the basic mechanistic details of the reactions catalyzed by each individual protein have been elucidated, relative.y little is known about how the two proteins interact with each other. In these proposed studies, structural and kinetic probes will be used to examine how these two enzymes functionally interact,, and then how the complex interacts with accessory proteins. The specific aims of this proposal can be summarized as follows; 1) Use photoactivateable crosslinking reagents to analyze the movement of the template during primase-catalyzed primer synthesis followed by pol alpha- catalyzed elongation of the primer. 2) Obtain a low resolution structure of pol alpha-primase using atomic force microscopy to determine the relative location of the DNA binding domains and 4 subunits of the complex. 3) Determine why the naturae of the primer (RNA versus DNA, primase synthesized versus exogenously added) can greatly alter the interactions of pol alpha with nucleotide analogs. 4) Elucidate how the single-stranded DNA binding protein RPA interacts with and alters the catalytic properties of pol alpha-primase. 5) Elucidate the roles of dNTPs and accessory proteins in determining where primase chooses to initiate synthesis. These studies will provide a detailed description of how new strands of DNA are initiated by pol alpha-primase. In turn, this will serve as a basis for elucidating how accessory proteins influence this reaction and, ultimately, how pol alpha-primase functions at the replication fork. In addition, these studies may answer why pol alpha interacts with nucleotide analogs in a fundamentally different manner when it elongates a primase synthesized primer compared to when it elongates a pre-existent primer. Since initiation of new strands of DNA presumably only occurs during DNA replication, understanding these unique properties may make it possible to take advantage of them and develop strategic to inhibit DNA replication without affecting other aspects of DNA metabolism.